The goal of my independent research program is to understand the mechanisms that control innate lymphoid cell (ILC) replenishment and differentiation in homeostasis and inflammation and how such regulation affects human health and disease. Experiments in this K22 proposal will generate essential preliminary data that lay the foundation of my independent research program. This proposal aims at discovering novel early ILC progenitors, exploring their importance in homeostasis and airway inflammation, and identifying the major molecules that drive early ILC development. Innate lymphoid cells are important players in host defense, tissue remodeling, and asthma pathogenesis. However, little is known about the cellular and molecular mechanisms that drive early ILC development. The transcriptional regulators TCF-1 and Id2 are required for the efficient generation of all known adult ILC subsets. I hypothesize that TCF-1 and Id2 are expressed at the earliest ILC progenitors and together drive early ILC development. Using new TCF-1gfp reporter mouse strain, I discovered a novel subset of early innate lymphoid cell progenitor (EILP) in the bone marrow and in the lung. EILP expressed TCF-1 and Id2, and efficiently differentiated into all four ILC lineages at the clonal level, indicating that they are the earliest identifiable ILC progenitor. I will use adoptive transfer and serial-transfer approaches to test whether EILP are the self-renewing progenitors that replenish non-circulating pulmonary ILC in homeostasis and during Alternaria-induced allergic airway inflammation. Using retroviral TCF-1 and Id2 co- transduction, I found that co-expression of both TCF-1 and Id2, but not either alone, elicited the emergence of ILC-committed progenitors in vitro. I will compare the transcriptomes of these TCF-1 and Id2 co-elicited progenitors with those of the in vivo early ILC progenitors to interrogate the gene program controlled by TCF-1 and Id2. I will further compare the transcriptomes of lung-resident EILP with those of bone marrow EILP and the in vitro TCF-1/Id2 co-elicited progenitors to explore whether lung microenvironments may elicit additional signals to support local ILC replenishment and differentiation. Together, these experiments will shed light on the hematopoiesis pathway that lead to ILC replenishment in the adult organism and will reveal the key gene regulatory pathways that drive early ILC development. The data generated will provide new insights into ILC biology and will suggest new therapeutic targets for ILC-related diseases such as asthma.